Disrupted cartilage products

ABSTRACT

This invention provides disrupted cartilage products, methods of manufacturing disrupted cartilage products, and methods of treating a subject comprising administering a cartilage product. The cartilage products are manufactured by a method comprising disrupting a collagen matrix, e.g. to produce a flexible cartilage product. Optionally, the cartilage products comprise viable chondrocytes, bioactive factors such as chondrogenic factors, and a collagen type II matrix. Optionally, the cartilage products are non-immunogenic.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to:

U.S. Provisional Application No. 61/670,434, entitled “DisruptedCartilage Products,” filed on Jul. 11, 2011 bearing Docket No. 25533US01in the office of McAndrews, Held and Malloy;

U.S. Provisional Application No. 61/670,424, entitled “Porated CartilageProducts,” filed on Jul. 11, 2011 bearing Docket No. 25532US01 in theoffice of McAndrews, Held and Malloy; and

U.S. Provisional Application No. 61/670,444, entitled “Methods ofManufacturing Cartilage Products,” filed on Jul. 11, 2011 bearing DocketNo. 25534US01 in the office of McAndrews, Held and Malloy; the contentsof which are hearby incorporated by reference in their entireties.

This application is being co-filed with a U.S. and PCT Applicationsentitled “Methods of Manufacturing Cartilage Products” bearing DocketNos. 25534US02 and 25534WO01 in the office of McAndrews, Held andMalloy, respectively; U.S. and PCT Applications entitled “PoratedCartilage Products” bearing Docket Nos. 25532US02 and 25532WO01 in theoffice of McAndrews, Held and Malloy, respectively; and PCT Applicationentitled “Disrupted Cartilage Products” bearing Docket No. 25533WO01 inthe office of McAndrews, Held and Malloy, which are hereby incorporatedby reference in their entirety.

TECHNICAL FIELD

The present invention relates to cartilage products useful intherapeutics and methods of producing and using such therapeutics.

BACKGROUND

Articular cartilage injury remains one of the major unsolved problems inorthopedics. Over 500,000 patients per year in the U.S. undergo surgicalprocedures to repair cartilage damage. However, many of these surgeriesyield suboptimal results.

Articular cartilage consists primarily of a sparse population ofchondrocytes distributed throughout an extracellular matrix formed byproteoglycans in a type II collagen fibril. The collagens give thetissue its form and tensile strength and the interaction ofproteoglycans with water give the tissue its stiffness to compression,resilience and durability. The hyaline cartilage provides a low frictionbearing surface over the bony parts of the joint. If the lining becomesworn or damaged resulting in lesions, joint movement may be painful orseverely restricted. Whereas damaged bone typically can regeneratesuccessfully, hyaline cartilage regeneration is quite limited.

Current surgical treatments include microfracture, debridement,osteochondral grafting, and autologous chondrocyte implantation (ACI).The goal of these treatments is to repair and regenerate native hyalinecartilage (collagen type II).

Microfracturing involves the removal of damaged articular cartilagefollowed by physically insulting the underlying subchondral bone toexposed bone marrow and create bleeding. Although the blood clotintroduces inflammatory cytokines, growth factors and MSCs to fill thedefect, the process fails to produce articular cartilage and insteadstimulates the production of fibrocartilage scar tissue, made fromcollagen type I. Fibrocartilage has poor long-term biomechanicalperformance, causes abnormal bone growth, and increases risk ofosteoarthritis.

Other strategies that have fallen short include autologous chondrocyteimplantation (ACI), debridement, and osteochondral grafting.

Gomes et al. (US 2004/0230303) describes an implant having a subchondralbone base and an articular cartilage cap containing bores drilledthrough the cartilage cap and base to allow cell migration. The implantcan be digested with hyaluronidase (type IV-s, 3 mg/mL) and trypsin(0.25% in monodibasic buffer 3 ml) for 18 hours at 37° C. Gomes et al.do not teach a flexible cartilage implant, a cartilage implantcontaining viable native chondrocytes, or a non-immunogenic cartilageimplant.

Chen et al. (US 2009/0024229) describes a cartilage graft that isdevitalized (made acellular) and then recellularized. The graft can bemicroperforated to facilitate recellularization. The graft can bedevitalized using enzymes to modify the molecular aspects of thecartilage such as chondroitinase to remove proteoglycan and arecombinant endonuclease, for example BENZONASE® (Merk, Inc.). Chen etal. do not teach a flexible cartilage implant or a cartilage implantcontaining viable native chondrocytes or a non-immunogenic cartilageimplant.

Steinwachs et al. (US 2008/0269895) describes cartilage implants havingvarious features. The implant can be a grown in-vitro from chondrocytesor can be a cartilage explant. Among various features, the implant canhave channels with a diameter of 0.5 mm to 2 mm. Among othershortcomings, Steinwachs et al. do not teach a digested cartilageimplant or a non-immunogenic cartilage implant.

Bardos et al. (“Osteochondral Integration of Multiply Incised PureCartilage Allograft: Repair Method of Focal Chondral Defects in aPorcine Model”; Am J Sports Med 2009 37: 50S) describes a pig cartilagesample comprising parallel incisions. Bardos et al. does not teach aspiral cut cartilage sample or a non-immunogenic cartilage sample.

Bravenboer et al. (“Improved cartilage integration and interfacialstrength after enzymatic treatment in a cartilage transplantationmodel”; Arthritis Res Ther 2004, 6) describes bovine articular cartilagetreated with hyaluronidase followed by collagenase. Among othershortcomings, Bravenboer et al. do not teach a mechanically disruptedcartilage sample or a non-immunogenic cartilage sample.

Bos et al. (“Specific Enzymatic Treatment of Bovine and Human ArticularCartilage”; Arthritis & Rheumatism Vol. 46, No. 4, April 2002, pp976-985) describes cartilage samples treated with collagenase VII. Boset al. does not teach a mechanically disrupted cartilage sample or anon-immunogenic cartilage sample.

What is needed in the art is a flexible cartilage product that can beeasily administered, for example, through an arthroscope cannula, andcontoured to a site of injured cartilage and provides a collagen type IImatrix containing viable chondrocytes and chondrogenic factors forregeneration of cartilage with minimal scarring.

SUMMARY OF THE INVENTION

The invention provides cartilage products, methods of manufacturing thecartilage products, and methods of using the cartilage products. Acartilage product according to the present invention comprises adisrupted collagen matrix. The collagen matrix comprises mechanicaldisruptions (e.g. collagen matrix is spiral cut), enzymatic disruptions(e.g. collagen matrix is collagenase-treated), or both mechanicaldisruptions and enzymatic disruptions.

The collagen matrix of exemplary cartilage products of the presentinvention have one or more (e.g. each) of the following technicalfeatures:

-   -   is derived from hyaline cartilage or comprises collagen II    -   comprises viable cells that are optionally native to the        collagen matrix;    -   comprises bioactive factors    -   exhibits greater flexibility than native articular cartilage;        and    -   is cryopreserved or formulated in a cryopreservation medium and        is substantially non-immunogenic.

In one embodiment, the collagen matrix comprises type II collagen (or“collagen II”). Optionally, a majority of the collagen in the collagenmatrix is collagen II, such as a collagen matrix provided by hyalinecartilage (e.g. articular cartilage). Optionally, the cartilage productis devoid of subchondral bone, calcified cartilage, or both subchondralbone and calcified cartilage. Optionally, the collagen matrix comprisesone or more cartilage layers selected from: a radial layer, atransitional layer, and a tangential layer.

In one embodiment, the cartilage product comprises viable cells.Optionally, the viable cells are chondrocytes. Optionally, the viablecells are native to the collagen matrix. Optionally, the viable cellsare distributed through the collagen matrix in a gradient. Optionally,the pores are aligned with the gradient.

In one embodiment, the cartilage product comprises bioactive factorssuch as chondrogenic factors. Optionally, the chondrogenic factors areselected from the group consisting of TGF-β1, TGF-β2, TGF-β3, BMP-2,BMP-7, bFGF, and IGF-1. Optionally, the cartilage comprises collagen II,hyaluronan, and aggrecan.

In one embodiment, the disruptions are enzymatic disruptions and the ECMprotein (e.g. collagen) fragments are substantially shorter than that ofnative articular cartilage. Additionally or alternatively, a substantialamount of the ECM protein in the collagen matrix is fragmented relativeto that of native articular cartilage. Optionally, the ECM proteinfragments are produced by enzymatic disruption (“digestion”) of a nativecartilage, e.g. digestion such as collagenase (e.g. Type II) treatment.

In one embodiment, the mechanical disruptions are of the tissue-removaltype. Optionally, the mechanical disruptions of the tissue-removal typeare selected from: spiral cuts, grooves, cross-grooves, pores, andhoop-forming core cuts.

In one embodiment, the mechanical disruptions are of thetissue-non-removal type. Optionally, the mechanical disruptions of thetissue-non-removal type are selected from: scores, spiral cuts,cross-scores, and piercings.

In one embodiment, the mechanical disruptions disrupt (i.e. extendthrough) the entire thickness of the cartilage sample. Alternatively,the mechanical disruptions disrupt less than the entirety of thethickness of the cartilage sample.

In one embodiment, the cartilage product is substantiallynon-immunogenic. Optionally, the cartilage product does not comprise asubstantial amount of macrophages.

In one embodiment, the collagen matrix has a thickness of about 0.5 toabout 2.0 mm. Optionally, the thickness is about 1 to about 2 mm.Optionally, the thickness is about 1 to about 1.5 mm.

In one embodiment, the disrupted collagen matrix is more flexible thanunmodified articular cartilage of similar thickness.

The invention also provides a method of making a cartilage productcomprising providing a cartilage sample and disrupting the cartilagesample by mechanical disruption, enzymatic disruption, or bothmechanical disruption and enzymatic disruption; and partially digestingthe cartilage sample.

Exemplary methods of manufacture of the present invention have one ormore (e.g. each) of the following technical features:

-   -   enzymatic disruption (“partial digestion”) is performed in a        manner that retains a substantial amount of viable cells;    -   the cartilage sample is mechanically disrupted to an extent that        increases flexibility of the cartilage sample;    -   the cartilage sample comprises hyaline cartilage; and    -   the cartilage sample is cryopreserved, e.g. after the step of        disruption.

In one embodiment, the step of partial digestion is performed in amanner that retains a substantial amount of viable cells, for example,by limiting digestion to a cell-sparing amount. Optionally, said step ofpartial digestion comprises digesting collagen. Optionally, said step ofpartial digestion comprises enzyme digestion, e.g. digestion with aproteinase such as collagenase (e.g. collagenase Type II).

In one embodiment, the cartilage sample is mechanically disrupted to anextent that increases flexibility of the cartilage sample. Optionally,the mechanical disruptions are provided which disrupt (i.e. extendthrough) the entirety of the thickness of the cartilage sample.Optionally, the mechanical disruptions are provided which do not disruptthe entirety of the thickness of the cartilage sample.

In one embodiment, the cartilage sample comprises hyaline cartilage.Optionally, the cartilage sample is an articular cartilage sample.Optionally, the cartilage sample is isolated from subchondral bone,calcified cartilage, or both.

In one embodiment, the cartilage sample is cryopreserved after the stepof disruption. Optionally, the step of cryopreservation comprisescryopreserving in a manner that spares viable cells.

In some embodiments, the cartilage products of the present invention arenot digested. In other embodiments, the cartilage products of thepresent invention are partially digested by digestive means includingenzymatic (e.g. collegenase, pronase, proteinase K, etc. treatment),biochemical (e.g. papain), thermal (e.g. increased heat), chemical(keratin sulfate, tosyllysylchloromethane), mechanical (perforated), anyother means of digestion known by those of skill in the art, andcombinations of any two or more of the foregoing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a knee joint from which a product of the presentinvention can be made.

FIG. 2 depicts layers of articular cartilage and adjacent bone of thepresent invention.

FIG. 3 depicts viable chondrocytes in cryopreserved cartilage productsof the present invention.

FIG. 4 depicts viable chondrocytes in cryopreserved cartilage productsof the present invention.

FIG. 5 depicts viable chondrocytes in cryopreserved cartilage productsof the present invention.

FIG. 6 depicts viable chondrocytes in cryopreserved cartilage productsof the present invention.

FIG. 7 depicts viable chondrocytes in cryopreserved cartilage productsof the present invention.

FIG. 8 depicts viable chondrocytes in cryopreserved cartilage productsof the present invention.

FIG. 9 depicts non-immunogenicity of cartilage products of the presentinvention.

FIG. 10 depicts sustained release of chondrogenic factors from cartilageproducts of the present invention.

FIG. 11 depicts greater release of chondrogenic factors from poratedcartilage products of the present invention.

FIG. 12 depicts greater release of chondrogenic factors from digestedcartilage products of the present invention.

FIG. 13 depicts greater release of chondrogenic factors fromcryopreserved cartilage products of the present invention.

FIG. 14 depicts the effect of iodine treatment on cell viability.

FIG. 15 depicts a cartilage product of the present invention. FIG. 15Adepicts the names used herein to reference surfaces of the cartilageproduct. FIG. 15 B illustrates the diameter used for surface areacalculation, the height used for thickness calculation, and the optionalorientation of layers or gradient.

FIG. 16 depicts cartilage products of the present invention comprising acollagen matrix and bone.

FIG. 17 depicts cartilage products of the present invention that havebeen subjected to mechanical disruption of the tissue-non-removal type.A) parallel scoring; B) non-aligned scoring; C) Spiral cut.

FIG. 18 depicts cartilage products of the present invention that havebeen subjected to mechanical disruption of the tissue-removal type. A)rectangular grooves, B) V-cut grooves (tapered inward); C) V-cut grooves(tapered outward); D) intersecting grooves.

FIG. 19 depicts cartilage products of the present invention that havebeen subjected to mechanical disruption of the hooping type. A) Singlehoop; B) Plurality of hoops.

FIG. 20 depicts a cartilage product of the present invention that hasbeen subjected to mechanical disruption of the spiral cut,tissue-removal type. A) Perspective view; B) Top view.

FIG. 21 depicts a cartilage product of the present invention that hasbeen subjected to mechanical disruption of the modified-spiral cut,tissue-removal type. A) Perspective view; B) Top view.

DETAILED DESCRIPTION OF THE INVENTION

As used here, the following definitions and abbreviations apply:

“BTB” means bone-tendon-bone graft obtained from the knee joint andcomprising the patella, the patellar tendon, and attached tibial boneblock.

“Cartilage product”, unless context demands otherwise, means a cartilageproduct of the instant invention.

“Disruption” means treating a collagen matrix in a manner that providesdiscontinuities in the matrix. In one embodiment, the disruptioncomprises enzymatic disruption (“partial disruption”). In anotherembodiment, the disruption comprises mechanical disruption. In anotherembodiment, the disruption comprises mechanical disruption and partialdigestion.

“DMEM” means Dulbecco's Modified Eagle Media.

“D-PBS” means Dulbecco's Phosphate Buffered Saline.

“ECM” means extracellular matrix, for example, the matrix of cartilage.

“Exemplary” (or “e.g.” or “by example”) means a non-limiting example.

“Mechanical disruption” means providing discrete discontinuities in thecollagen matrix by mechanical treatment. The discrete discontinuitiescan be provided by tissue-removal technique (e.g. as depicted in FIG. 18through FIG. 21) or by non-tissue-removal technique (e.g. as depicted inFIG. 17). In one embodiment, the discrete discontinuities are providedthrough the entire thickness of the collagen matrix (e.g. as depicted inFIG. 17B, FIG. 17C, and FIG. 19 through FIG. 21). In another embodiment,the discrete discontinuities are provided through less than the entirethickness of the collagen matrix (e.g. as depicted in FIG. 17A and FIG.18). In one embodiment, a mechanically disrupted collagen matrix doesnot comprise a circular cartilage disk comprising a removed core (e.g.as depicted in FIG. 19A) if the removed core has a surface area of lessthan 15% compared to the cartilage disk before said core removal.

“Natural”, in the context of, for example, “natural ECM” or “naturalcartilage”, refers to properties exhibited by the ECM or cartilage inits natural state in the donor.

“Partial digestion” (or “limited digestions”) means enzymatic digestionwherein one or more digestible sites remain un-digested. In oneembodiment, partial digestion is a cell-sparing digestion such thatfurther digestion otherwise decreases cell viability. In one embodiment,digestion can be monitored by any method, e.g. measuring the release ofdigestion products from a cartilage sample or by the effect of digestionon the physical properties. In one embodiment, a partially digestedcollagen matrix (e.g. articular cartilage sample) is substantiallyintact relative to an undigested collagen matrix, for example, thedigested collagen matrix retains its shape throughout digestion.

“QC” means Quality Control

“Substantial amount” when used with respect to therapeutic cells (e.g.chondrocytes) and therapeutic bioactive factors (e.g. chondrogenicfactors) in a cartilage product means an amount which provides ameasurable therapeutic effect in vivo when the cartilage product isadministered, e.g. according to the present treatment methods.

The term “devoid” of a substance as used with respect to the presenttechnology includes products that are “substantially free of” or“substantially devoid of” such substance, and includes products thathave less than 5% of the substance, more preferably less than 2%, morepreferably less than 1%, more preferably less that 0.5%, including 0% ofsuch substance. For example, in some embodiments of the presentinvention, devoid of subchondral bone, calcified cartilage, or bothsubchondral bone and calcified cartilage when used with respect to thepresent technology includes cartilage products which are substantiallyfree of subchondral bone, calcified cartilage or both, cartilageproducts which are substantially devoid of subchondral bone, calcifiedcartilage or both, and products that contain less than 5%, less than 2%,less than 1%, less than 0.5% or 0% of subchondral bone, calcifiedcartilage or both.

Cartilage Products

The present invention provides a cartilage product comprising a collagenmatrix having plurality of pores therein. Optionally, the collagenmatrix comprises ECM protein fragments, e.g. the collagen matrix isdigested hyaline cartilage.

In some embodiments, the cartilage product comprises a matrix having oneor more pores therein.

Surprisingly, exemplary cartilage products of the present inventionsupport the regeneration of healthy normal articular cartilage byproviding type II collagen and proteoglycans, bioactive factors, andviable chondrocytes.

In one embodiment, the cartilage product is flexible. An exemplaryflexible cartilage product can be rolled into an arthroscope cannula,can bend extensively without breaking, and can contour to irregulartarget sites in a subject. Surprisingly, it has been discovered that aflexible cartilage product with viable cells and factors can be producedby appropriately configuring a) the thickness of the collagen matrix; b)extent of mechanical disruption; and c) the extent of digestion.

In one embodiment, the cartilage product comprises viable cells such aschondrocytes. Optionally, the viable cells are native chondrocytes.Optionally, the viable cells are distributed through the collagen matrixin a gradient. Optionally, the collagen matrix comprises mechanicaldisruptions extending at least partially through the gradient (e.g.pores provided that are substantially aligned with the gradient orperpendicular to the superior surface, as depicted in FIG. 15).

In one embodiment, the collagen matrix is enzymatically disrupted andthe ECM protein (e.g. collagen) fragments are substantially shorter thanthat of native articular cartilage. Additionally or alternatively, asubstantial amount of the ECM protein in the collagen matrix isfragmented relative to that of native articular cartilage. Optionally,the ECM protein fragments are produced by partial digestion of a nativecartilage, e.g. enzymatic digestion such as collagenase (e.g. Type II)treatment.

In one embodiment, the cartilage product is formulated forcryopreservation, e.g. comprises a cryopreservation medium. Optionally,the cartilage product is cryopreserved.

To illustrate one embodiment of the invention, an exemplary cartilageproduct comprises, as the collagen matrix, a layer (e.g. disk) ofhyaline (e.g. articular) cartilage having mechanical disruptions (e.g.spiral cut), wherein the layer of cartilage comprises viable nativechondrocytes and collagen fragments produced by partial digestion with acollagen-digesting enzyme such as a collagenase. The layer of cartilageis flexible while retaining its structural integrity. The cartilageproduct is devoid of subchondral bone and calcified cartilage andcomprises a radial layer, a transitional layer, and a tangential layerthroughout which a gradient of the viable native chondrocytes aredistributed. Optionally, the cartilage product is formulated forcryopreservation.

Collagen Matrix

A cartilage product of the present invention comprises a collagenmatrix. The collagen matrix can be any extracellular matrix comprisingcollagen fibrils and bioactive factors. The collagen matrix can beobtained from any source and can be any size and shape. Optionally, thecollagen matrix is flexible (e.g. such that it can be rolled or foldedand administered via arthroscope cannula).

In one embodiment, the collagen matrix isolated from a subject (‘naturalcollagen matrix’) or is grown in-vitro.

In one embodiment, the collagen fibrils comprise type II collagen.Optionally, the collagen matrix is hyaline cartilage such as articularcartilage. Optionally, the articular cartilage is condoyle cartilage,femur condoyle cartilage, tibial plateau cartilage, femoral headcartilage, humoral head cartilage, talus cartilage, or acetabulumcartilage. Optionally, the articular cartilage comprises one or morelayers of cartilage selected from: a radial layer, a transitional layer,and a tangential layer.

In one embodiment, the collagen matrix comprises fragmented ECMproteins. Fragmented ECM proteins are optionally produced by partialdigestion of a natural collagen matrix. Optionally, the collagen matrixis partially digested with a proteinase such as a collagen-degradingenzyme. Useful collagen-degrading enzymes include, but are not limitedto, for example, collagenase (e.g. Types I-IV, bacterial collagenase),other endopeptidases (e.g. trypsin, papain, pepsin), and exopeptidases(e.g. carboxypeptidase).

In one embodiment, the collagen matrix is devoid of subchondral bone,calcified cartilage, or both. If present, subchondral bone and calcifiedcartilage can otherwise inhibit the flexibility of the collagen matrix.In an alternative embodiment, the cartilage product comprises bone, e.g.a reduced-area plug of bone (relative to the collagen matrix) asdepicted in FIG. 16A or a shell of bone as depicted in FIG. 16B.

In one embodiment, the collagen matrix is derived from articularcartilage and comprises one or more layers of cartilage selected from: aradial layer, a transitional layer, and a tangential layer. As depictedin FIG. 2, native chondrocytes of a natural articular cartilage aredistributed across these layers in a gradient from vertical rows ofchondrocytes in the radial layer to flattened cells in the tangentiallayer. Collagen fibrils of the tangential (‘superficial’) layer runparallel to the surface. Collagen fibrils in the radial layer aretypically oriented towards (e.g. perpendicular to) the articularsurface. Collagen fibers in the transitional layer are typically lesspacked than that of the radial and tangential layers and arrangedobliquely or in a more randomized fashion to the articular surface.

In one embodiment, the collagen matrix has a thickness (or ‘height’ asdepicted in FIG. 15) of less than about 3 mm. Optionally, the collagenmatrix has a thickness of about 0.2 mm to about 2 mm or about 1 mm toabout 1.5 mm. The thickness can be measured, for example, perpendicularto layers of the collagen matrix (e.g. distance from the surface of atangential layer to the surface of a radial layer), as depicted in FIG.15A. Surprisingly, collagen matrices of reduced thickness and tailoredmechanical disruption can provide a flexible cartilage product that canbe administered by arthroscopy and easily contoured to an injured tissuesite while retaining its capacity to provide a matrix of viable cellsand factors.

In one embodiment, the collagen matrix has a surface area (e.g. superioror inferior surface, such as the surface of a tangential layer or aradial layer, respectively) having an area of about 0.5 cm² to about 5cm². Optionally, the collagen matrix has a superior surface and aninferior surface separated by a thickness of less than about 3 mm orless than about 2 mm (e.g. 1 mm to about 1.5 mm).

In one embodiment, the collagen matrix is provided in a round shape(e.g. oval or circle), a rectangular shape, or a square shape. Anexample of a cartilage product comprising a round collagen matrix isdepicted in FIG. 15. Optionally, the width (referred to herein as‘diameter’ regardless of shape) of a collagen matrix is greater than theheight (referred to herein as ‘thickness’), e.g. as depicted in FIG. 15.

In one embodiment, the collagen matrix is a disk, i.e. the diameter (orwidth) of the collagen matrix surface (e.g. superior or inferiorsurface) is greater than the thickness (or height of the lateralsurface) of the collagen matrix, e.g. as depicted in FIG. 15.Optionally, the pores are provided in the superior surface, the inferiorsurface, or both.

In one embodiment, relative to an unmodified collagen matrix (e.g.isolated articular cartilage sample) the collagen matrix has enhancedflexibility (e.g. reduced shear modulus) but retains mechanicalproperties of cartilage. Examples of such properties (e.g. compressionstrength, Young's modulus) are described in MANSOUR (“Biomechanics ofCartilage” Ch 5; Obtained from the internet URL:http://www.cartilagehealth.com/images/artcaribiomech.pdf)

In one embodiment, the collagen matrix is flexible (e.g. such that itcan be folded without breaking) and comprises a Young's modulus of atleast about any of: 0.1 MPa, 0.2 MPa, 0.3 MPa, or 0.4 MPa.

Mechanical Disruptions

In one embodiment, a cartilage product of the present inventioncomprises mechanical disruptions. The mechanical disruptions can be anydiscrete discontinuities in the collagen matrix.

In one embodiment, the discrete discontinuities are of thetissue-removal type (e.g. as depicted in FIG. 18 through FIG. 21).Useful mechanical disruptions of the tissue-removal type include: spiralcuts, grooves, cross-grooves, radial cuts, pores, and hoop-forming corecuts (e.g. single or multiple hoop forming cuts). In another embodiment,the discrete discontinuities are of the tissue-non-removal type (e.g. asdepicted in FIG. 17). Useful mechanical disruptions of thetissue-non-removal type include: scores (e.g. as in FIG. 17A), spiralcuts, cross-scores, radial scores (e.g. as in FIG. 17B), and piercings(e.g. a plurality of piercings imparted with a thin, cylindrical,pointed object).

In one embodiment, the discrete discontinuities are provided through theentire thickness of the collagen matrix (e.g. as depicted in FIG. 17B,FIG. 17C, and FIG. 19 through FIG. 21). In another embodiment, thediscrete discontinuities are provided through less than the entirethickness of the collagen matrix (e.g. as depicted in FIG. 17A and FIG.18).

In one embodiment, the mechanical disruptions are partial mechanicaldisruptions, i.e. the collagen matrix substantially retains its shaperelative to that of an unmodified collagen matrix (e.g. as depicted inFIG. 15 through FIG. 21). In another embodiment, the mechanicaldisruptions are complete disruptions (e.g. the collagen matrix isminced, e.g. into a plurality of solid pieces, into a viscous form, orinto an amorphous form).

Spiral Cut

In one embodiment, a cartilage product of the present inventioncomprises a spiral cut collagen matrix. The spiral cut can be anycontinuous cut that encircles or otherwise circumnavigates the collagenmatrix and comprises greater than one full turn. Optionally, the spiralcut extends through the entire thickness of the collagen matrix, e.g. asdepicted in FIG. 17, FIG. 20 and FIG. 21

In one embodiment, the spiral cut is provided of the tissue-non-removaltype, e.g. as depicted in FIG. 17. In another embodiment, the spiral cutis of the tissue-removal type, e.g. as depicted in FIG. 20 and FIG. 21.Optionally, spiral cut collagen matrices of the tissue-non-removal typecan construct to fit in a void (e.g. removed cartilage defect in apatient) that is substantially smaller in area (e.g. 90% or less)relative to the collagen matrix (e.g. a spiral cut collagen matrix diskwith a diameter of 2 cm is configured such that it can be squeezed andconstricted down to a diameter of about 1.8 cm).

In one embodiment, the spiral cut comprises at least two turns, at leastthree turns, at least four turns, or at least five turns. For example,FIG. 17, FIG. 20 and FIG. 21 depict collagen matrices comprising aspiral cut with three turns.

In one embodiment, the spiral cut is of the unbranched type (e.g. asdepicted in FIG. 20) or the branched type (e.g. as depicted in FIG. 21).

Pores

In one embodiment, a cartilage product of the present inventioncomprises a collagen matrix having a plurality of pores. The pluralityof pores can be configured in any manner that increases the flexibilityof the cartilage and provides a plurality of passageways through whichcells and factors can migrate.

The pores can be provided through any surface of the collagen matrix andcan extend partially (as in a cavity) or entirely (as in a channel)through the collagen matrix. For example, as depicted in FIG. 15, thepores can be provided through a superior surface, an inferior surface,or both.

According to the present invention, pores in the collagen matrix provideone or more of the following technical features:

-   -   a flexible cartilage product;    -   a cartilage product that has superior fixation; and    -   a cartilage product that facilitates migration of mesenchymal        stem cells (MSCs) and chondrocytes

In one embodiment, plurality of pores is configured to impartflexibility to the collagen matrix. By varying the density and size ofthe pores, one skilled in the art can produce a flexible collagen matrixaccording to the present invention. Optionally, the plurality of poreshave a diameter of about 0.25 mm to about 1.5 mm such as about 0.5 mm toabout 1.5 mm (e.g. about 1 mm). Optionally, the collagen matrixcomprises a pore density of about 10 to about 500 pores per cm² such asabout 10 to about 100 pores per cm² or about 10 to about 60 pores percm² (e.g. about 36 pores per cm²). Optionally, the collagen matrixcomprise a superior or inferior surface having a total pore area fromabout 3% to about 90% (e.g. about 5% to about 50% or about 10% to about50%) relative to the total area of said surface. For example, a collagenmatrix having pores with a diameter of about 1 mm at 36 pores per cm²has a pore area of about 28% relative to that of the total area of thesurface.

The plurality of pores optionally imparts improved fixation. Pores canincrease the surface area of the collagen matrix and provide betteradhesion, for example, facilitating fixation upon application of anadhesive (e.g. fibrin glue such as Tisseel) or allowing greaterintegration into the target site in a subject to which the cartilageproduct is optionally administered.

The plurality of pores optionally facilitates migration of MSCs andchondrocytes (e.g. donor cells endogenous to the collagen matrixmigrating out of the collagen matrix and cells of the implant recipientmigrating into the collagen matrix).

In one embodiment, the pores are channels or cavities. A channel is anypore that extends through two faces (e.g. through a superior surface andan inferior surface) of the collagen matrix. A cavity is any pore thatdoes not extend through two faces of the collagen matrix. Optionally,the pores are arranged in an array, e.g. a two dimensional array.Optionally, the collagen matrix comprises one or more cellular or ECMlayers (e.g. radial, tangential, or transitional layers) and the poresextend substantially perpendicularly (i.e. 90±45°) to the layer(s) ordiagonally to the layer(s), or the collagen matrix comprises a gradientof cells and the pores are aligned or substantially parallel (i.e.0±45°) with the gradient, e.g. as depicted in FIG. 15.

In one embodiment, the collagen matrix comprises a plurality of poreshaving a diameter selected from: about 0.3 mm to about 2 mm, about 0.5mm to about 1.5 mm, about 0.8 mm to about 1.2 mm, or about 1 mm.

In one embodiment, the collagen matrix comprises a plurality of pores,wherein about 3% to about 90% of the surface area is porated (e.g. about3% to about 50% or about 5% to about 50% or about 3% to about 30% orabout 5% to about 50%). For example, a collagen matrix comprisingcylindrical pores with a 1 mm diameter at a pore density of 36 pores percm² would comprise about 28 mm² of porated surface area per to cm² oftotal surface area of the collagen matrix [(0.5 mm pore radius)×(π)×(36pores/cm²)], i.e. 28% porated surface area

In one embodiment, the pore size is about 50% to about 150% of thethickness of the collagen matrix or pore length.

Pores can be produced in any manner, for example, mechanical removal ofcollagen matrix using a drill or tissue punch.

Fragmented ECM

According to the present invention, enzymatic disruption (“partialdigestion”) is optionally provided in a cartilage product to providefragmented ECM proteins.

In one embodiment, the fragmented ECM proteins are collagen (e.g. TypeII) fragments or proteoglycan fragments. Optionally, the collagen matrixis articular cartilage.

Fragmented ECM proteins can be produced in any manner. Fragmented ECMproteins are optionally produced by partial digestion of a naturalcollagen matrix (i.e. isolated from a subject). Optionally, the collagenmatrix is partially digested with a digestion enzyme (e.g. proteinase)such as, for example, a collagen-degrading enzyme (e.g. collagenase) ora proteoglycan-degrading enzyme (e.g. hyaluranidase).

According to the present invention, partial digestion of a collagenmatrix provides one or more of the following technical features:

-   -   a loose ECM that releases and allows migration of cellular        factors and viable cells.    -   a natural ECM retaining viable native cells    -   preservation of physiologic interactions between cells and the        ECM    -   a clean cartilage product devoid of debris    -   a cartilage product comprising ECM fibrils that substantially        retain the packing density of natural cartilage    -   a cartilage product with greater flexibility    -   removes macrophages and reduces immunogenicity

A fragmented ECM provides a loose collagen matrix that releases andallows migration of cellular factors and viable cells. For example,cellular (e.g. chondrogenic) factors can leach out into the surroundingmicro environment upon administration to a subject. With the teachingsprovided herein, one skilled in the art can now tailor digestion toprovide such a technical feature.

In one embodiment, the fragmentation of ECM is limited to an amount thatretains a substantial amount of viable native cells. Upon furtherfragmentation, the collagen matrix can prematurely release itspopulation of cells before optional administration of the cartilageproduct. With the teachings provided herein, one skilled in the art cannow tailor digestion to provide such a technical feature.

In one embodiment, the ECM is fragmented in a manner that preservesnormal interactions between cells and the ECM. For example, the ECMand/or cellular factors therein activate chondrocytes, i.e. induce ashift from the G0 phase to the G1 phase, and also induce MSCs toinfiltrate and differentiate into chondrocytes. Without being bound bytheory, the inventors believe that these functions enhance therapeuticefficacy.

In one embodiment, the ECM is fragmented in a manner that preservesnormal interactions between bioactive factors and the ECM. For example,bioactive factors are retained at levels to greater than about 50% orgreater than about 70% compared to predigestion levels.

In one embodiment, the ECM is fragmented in a manner that cleans thecartilage sample of debris. This microscopic and/or macroscopic debris(e.g. ECM fragment) is present in even greater amounts upon poration ofa cartilage sample, and can trigger pain and other adverse responseswhen administered to a subject if the cartilage sample of the presenttechnology is not cleansed of debris.

In one embodiment, fragmentation of the ECM is limited to an amount thatprovides fragmented ECM fibrils that substantially retain the packingdensity of natural cartilage. Such a technical feature provides acartilage product having mechanical properties of native cartilage.

In one embodiment, fragmentation of the ECM is limited to an amount thatprovides a collagen matrix that has any (e.g. each) of the followingtechnical features: is visually intact, is flexible (e.g. such that itcan be folded or bent without breaking or rolled to fit in anarthroscope), retains viable native cells, retains non-degradedbiofactors (e.g. growth factors), and increases the level of biofactors(e.g. growth factors).

In one embodiment, the cartilage product exhibits greater flexibilitywith the fragmented ECM compared to that of the same collagen matrixwithout fragmented ECM. Optionally, the cartilage product is flexiblesuch that it can be inserted into a cannula having a diameter not morethan 50% of the diameter (or width) of the cartilage product. Forexample, the cartilage product in the shape of a disk with a diameter of2 cm can be flexible enough such that it can be rolled into a cannula ofan arthroscope (e.g. a cannula with a diameter of less than about 1 cm).

Viable Chondrocytes

In one embodiment, a cartilage product of the present inventioncomprises viable chondrocytes. Optionally, the cartilage product is anatural cartilage product (i.e. the collagen matrix is isolated from asubject) and the viable chondrocytes are native, i.e. native to thecollagen matrix. Viability can be demonstrated by any means, e.g.through the use of vital stains, phase contrast microscopy, etc.

In one embodiment, the collagen matrix is derived from articularcartilage and comprises viable chondrocytes that are native (orendogenous) to the articular cartilage. Native chondrocytes aredistributed across one or more (e.g. all) layers of articular cartilageselected from: a radial layer, a transitional layer, and a tangentiallayer. The invention alternatively contemplates collagen matrices havingexogenous or non-native (i.e. added) chondrocytes. In one embodiment,the collagen matrix comprises viable chondrocytes at its surface.Optionally, at least 70% of the chondrocytes at the surface of thecollagen matrix are viable. In some embodiments, the cartilage productcomprises at least about 50% viable cells, alternatively at least 60%viable cells. In some embodiments, the cartilage product comprises atleast about 70% viable cells, alternatively about 75% viable cells,alternatively about 80% viable cells.

Surface viable cell percentage can be quantified, for example, bymicroscopy techniques.

In one embodiment, a portion of the chondrocytes are in the G₀ phase.Without being bound by theory, the inventors believe that the ECM or ECMfactors activate chondrocytes. This activation can be observed as ashift to the G₁ phase from G₀.

Chondrocytes are thought to be important in maintaining cartilage matrixhomeostasis in addition to expressing factors that promotechondrogenesis and cartilage repair. Without being bound by theory, theinventors believe that a superior therapeutic product is obtained bypreservation of the cellular and structural organization of nativearticular cartilage.

Without being bound by theory, the inventors believe that the collagenmatrix of cartilage products of the present invention preserves theviability of chondrocytes and extends their life-span ex-vivo (includingin the recipient subject). In addition, it is believed that, uponadministration to a recipient subject, the collagen matrix can inducethe recipient's MSCs to infiltrate the collagen matrix (of the cartilageproduct) and differentiate into chondrocytes, thereby replenishing thecartilage product with chondrocytes.

As detailed in Example 10, cartilage products of the present inventioncan contain a substantial amount of viable chondrocytes, even afterpartial digestion and cryopreservation. A substantial amount of viablechondrocytes is an amount which, when present, enhances the therapeuticefficacy of a cartilage product.

Bioactive Factors

In one embodiment, a cartilage product of the present inventioncomprises bioactive factors. Optionally, bioactive factors comprisechondrogenic factors. Optionally, the chondrogenic factors include oneor more (e.g. each) of TGF-β1, TGF-β2, TGF-β3, BMP-2, BMP-7, bFGF, andIGF-1.

Optionally, the cartilage comprises an extracellular matrix comprisingcollagen type II, hyaluronan, and aggrecan.

Optionally, the cartilage comprises transcription factors, e.g. Sox5,Sox6, and Sox9.

In one embodiment, the cartilage product comprises TGF-β1, TGF-β2,TGF-β3, BMP-2, BMP-7, bFGF, IGF-1, collagen type II, hyaluronan,aggrecan, Sox5, Sox6, and Sox9.

In one embodiment, the collagen matrix is a natural collagen matrix andthe bioactive factors are native to the collagen matrix.

As detailed in Example 16, cartilage products of the present inventioncan contain bioactive factors (e.g. chondrogenic factors), even afterpartial digestion and cryopreservation. Additionally or alternatively,cartilage products of the present invention can release bioactivefactors when cultured in vivo or in vitro. The amount of a given factorin cartilage product can be determined by a tissue lysate assay, e.g. asdetailed in Example 16. The amount of factor released from a cartilageproduct can be determined by a culture assay, e.g. as detailed inExample 16. For example, as detailed in Example 16 and Table 4, acartilage product of the present invention can have one or more (e.g.each) of the following technical features:

-   -   a. comprises TGF-β1 in an amount of at least about 11 pg/cm²,        for example, about 11 to about 628 pg/cm².    -   b. comprises TGF-β3 in an amount of at least about 4 pg/cm², for        example, about 4 to about 112 pg/cm².    -   c. comprises BMP-7 in an amount of at least about 3 pg/cm², for        example, about 3 to about 23 pg/cm².    -   d. comprises bFGF in an amount of at least about 169 pg/cm², for        example, about 169 to about 365 pg/cm².    -   e. comprises IGF-1 in an amount of at least about 111 pg/cm²,        for example, about 111 to about 779 pg/cm².    -   f. when cultured, the cartilage product releases TGF-β1 in an        amount of at least about 2617 pg/cm², for example, about 2617 to        about 17818 pg/cm².    -   g. when cultured, the cartilage product releases TGF-β2 in an        amount of at least about 133 pg/cm², for example, about 133 to        about 623 pg/cm².    -   h. when cultured, the cartilage product releases IGF-1 in an        amount of at least about 14 pg/cm², for example, about 14 to        about 2842 pg/cm².

Without being bound by theory, the inventors believe that important toefficient cartilage repair, as facilitated by cartilage products, aregrowth factors, chondrogenic factors, and other bioactive factors whichmediate extracellular matrix production and promote chondrogenesis invivo. For example, TGF-β 1-3 promote chondrogenic differentiation andregulate collagen expression; BMP-2 and BMP-7 Induce chondrogenesis ofMSCs and stimulate ECM production by chondrocytes; bFGF stimulatesproliferation of chondrocytes; IGF-1 induces ECM synthesis; and ECM(Collagen, Hyaluronan, and Aggrecan) mediates mechanical regulation ofchondrogenesis.

Formulation

According to the present invention, the cartilage product is optionallyformulated with a cryopreservation medium.

In one embodiment, the cryopreservation medium comprising one or morecell-permeating cryopreservatives, one or more non cell-permeatingcryopreservatives, or a combination thereof.

Optionally, the cryopreservation medium comprises one or morecell-permeating cryopreservatives selected from, but not limited to, forexample, DMSO, a glycerol, a glycol, a propylene glycol, an ethyleneglycol, or a combination thereof.

Optionally, the cryopreservation medium comprises one or more noncell-permeating cryopreservatives selected from, but not limited to, forexample, polyvinylpyrrolidone, a hydroxyethyl starch, a polysacharide, amonosaccharides, a sugar alcohol, an alginate, a trehalose, a raffinose,a dextran, or a combination thereof.

Other examples of useful cryopreservatives are described in“Cryopreservation” (BioFiles Volume 5 Number 4-Sigma-Aldrich®datasheet).

In one embodiment, the cryopreservation medium comprises acell-permeating cryopreservative, wherein the majority of thecell-permeating cryopreservative is DMSO. Optionally, thecryopreservation medium does not comprise a substantial amount ofglycerol.

In one embodiment, the cryopreservation medium comprises DMSO, e.g. inan amount of about 1% to about 50% DMSO by volume (e.g. about 10%).

In one embodiment, the cryopreservation medium comprises additionalcomponents such as albumin (e.g. HSA or BSA), an electrolyte solution(e.g. Plasma-Lyte, PBS, or saline), or a combination thereof.

In one embodiment, the cryopreservation medium comprises 1% to about 20%albumin (e.g. HSA) by weight and about 1% to about 50% cryopreservativeby volume (e.g. about 10%) such as DMSO.

Non-Immunogenicity

In one embodiment, the cartilage product is substantiallynon-immunogenic.

Cartilage products of the present invention have one or more technicalfeatures that reduce immunogenicity. Examples of such technical featuresinclude:

-   -   Absence of non-sequestered cells    -   Presence of immunoprivileged MSC cells and low levels of        circulating immunogenic cells (e.g. macrophages) and TNF-α.    -   Selective killing by cryopreservation

As taught herein, certain embodiments of the present invention comprisea collagen matrix having viable cells such as chondrocytes that arenative to the collagen matrix. The invention also contemplates cartilageproducts having non-native cells such as chondrocytes added to thematrix. While exogenous (and endogenous) chondrocytes are a potentialsource of immunogenicity, cartilage products of the present inventionsurprisingly exhibit low or absent immunogenicity. Without being boundby theory, the inventors believe that chondrocytes (especially nativechondrocytes), which are embedded in the collagen matrix, are moreeffectively sequestered from the surrounding environment in a subject towhich the cartilage product is administered, thereby reducingimmunogenicity.

In one embodiment, the cartilage product has depleted levels ofcirculating immunogenic cells and TNF-α. Optionally, such a cartilageproduct substantially lacks a response to lipopolysaccharide (LPS). Sucha cartilage product can be provided, for example, by performingmanufacturing steps of washing/rinsing, digestion, cryopreservation, orany combination thereof.

Manufacture

A cartilage product can be produced in any manner. In one embodiment,the invention provides a method of making (“manufacturing”) a cartilageproduct comprising providing a cartilage sample and disrupting thecartilage sample. The step of disrupting the cartilage sample comprisemechanical disruption, enzymatic disruption (“partial digestion”), orboth mechanical disruption and partial digestion.

In one embodiment, the method comprises removing from the cartilagesample subchondral bone, calcified cartilage, or both subchondral boneand calcified cartilage.

In one embodiment, the method comprises cryopreserving the cartilagesample after said steps of porating and partial digestion. Optionally,the step of cryopreservation comprises cryopreserving in a manner thatspares viable cells.

In one embodiment, the step of partial digestion is performed in amanner that retains a substantial amount of viable cells.

In one embodiment, the cartilage sample is mechanically disrupted to anextent that increases flexibility of the cartilage sample.

In one embodiment, processing of the cartilage sample is performed in amanner that does not generate a substantial amount of heat. Optionally,cutting of cartilage tissue comprises the use of a low-speed saw ordrill and/or a tissue punch.

In one embodiment, the process comprises chilling (e.g. continually orperiodically) the cartilage sample.

Cartilage Sample

The cartilage sample can be obtained from any source and can be providedin any shape, thickness, and surface area. Optionally, the source is asubject such as a human subject. Optionally, the source is a cadaver.

In one embodiment, the cartilage sample is any cartilage samplecomprising type II collagen. Optionally, the cartilage sample isselected from: hyaline cartilage, fibrocartilage, and elastic cartilage.

In one embodiment, the cartilage sample comprises hyaline cartilage.Optionally, the cartilage sample is an articular cartilage sample (e.g.obtained from a donor bone). Optionally, the cartilage sample isisolated (i.e. separated) from subchondral bone and/or calcifiedcartilage. Cartilage can be separated from subchondral bone afterremoving a cartilage sample in the form of an osteochondral plug (e.g.using a tissue punch) or the cartilage can be separated directly fromsubchondral bone while present on the donor bone (e.g. by slicing offcartilage from the donor bone). Other useful hyaline cartilages includenasal cartilage, tracheal cartilage, and laryngeal cartilage

In one embodiment, the cartilage sample is articular cartilage.Articular cartilage can be obtained from any donor bone. Optionally, thecartilage sample is obtained from long bones such as femur, tibia,fibula, humerus, ulna, radius, or short bones such as the bones of thehands or feet (e.g. talus), flat bones such as pelvic bones (e.g.acetabulum), irregular bones such as vertebrae, and sesamoid bones.Articular cartilage can be obtained from the condoyle of any bone.Optionally, the cartilage sample is obtained as a plug (e.g. 1 cm or 2cm plug). Optionally, the cartilage sample is removed of subchondralbone and/or calcified cartilage.

In one embodiment, the cartilage sample comprises fibrocartilage.Optionally, the cartilage sample is obtained from a source selectedfrom: pubic symphysis, annulus fibrosis, intervertebral disc, meniscus,and temporomandibular joint.

In one embodiment, the cartilage sample comprises elastic cartilage.Optionally, the cartilage sample is obtained from a source selected fromears, larynx, epiglottis.

In one embodiment, the cartilage sample is obtained from a mammal, anungulate, an organism of the Sus genus, a pig, a primate, a higherprimate, or a human.

In one embodiment, the cartilage sample is screened for thickness.Optionally, the thickness is 0.2 mm to about 2.0 mm such as about 1 mmto about 1.5 mm. For example, cartilage samples that are thinner thanthe minimum thickness can be discarded while cartilage samples that arethicker than the maximum thickness are trimmed down to size.Surprisingly, by reducing the thickness of the cartilage sample andporating the cartilage sample, a flexible cartilage product is obtainedthat easily contours to an injured tissue site while retaining thecapacity to provide a matrix of viable cells and bioactive factors.

In one embodiment, the collagen sample has a surface (e.g. upper(“superior”) or lower (“inferior”) surface) having an area of about 0.5cm² to about 5 cm². Optionally, the collagen sample has an upper surfaceand a lower surface separated by a thickness of less than about 2 mm(e.g. 1 mm to about 1.5 mm).

In one embodiment, the collagen sample is provided in a round shape(e.g. oval or circle), a rectangular shape, or a square shape.

In one embodiment, obtaining the cartilage sample comprises chilling thesample, e.g. using chilled solvent, a cold room, a cold plate. In oneembodiment, obtaining the sample comprises isolating without generatinga substantial amount of heat, e.g. using a low speed saw. In someembodiments, chilling comprises the use of an ice water bath.

Mechanically Disrupting

A method of producing a cartilage product of the present inventionoptionally comprises a step of mechanically disrupting the cartilagesample. The step of mechanical disruption can be performed in any mannerthat imparts discrete discontinuities in the collagen matrix.

Useful methods of mechanical disruption include: cutting (e.g. scoringwithout removing tissue or cutting with tissue removal), grooving,porating, piercing, and mincing.

In one embodiment, the mechanical disruption comprises removing tissue(e.g. products depicted in FIG. 18 through FIG. 21). Useful mechanicaldisruptions of the tissue-removal type include: spiral cuts, grooves,cross-grooves, pores, and hoop-forming core cuts. In another embodiment,the mechanical disruption comprises providing discontinuities withoutremoving a substantial amount of tissue (e.g. as depicted in FIG. 17).Useful mechanical disruptions of the tissue-non-removal type include:scores, spiral cuts, cross-scores, and piercings.

In one embodiment, the mechanical disruption is performed in a mannerthat provides discrete discontinuities through the entire thickness ofthe collagen matrix (e.g. as depicted in FIG. 17B, FIG. 17C, and FIG. 19through FIG. 21). In another embodiment, the mechanical disruption isperformed in a manner that provides discrete discontinuities throughless than the entire thickness of the collagen matrix (e.g. as depictedin FIG. 17A and FIG. 18).

In one embodiment, the mechanical disruptions are partial mechanicaldisruptions, i.e. the collagen matrix substantially retains its shaperelative to that of an unmodified collagen matrix (e.g. as depicted inFIG. 15 through FIG. 21).

In another embodiment, the mechanical disruptions are completedisruptions (e.g. the collagen matrix is minced, e.g. into a pluralityof solid pieces, into a viscous form, or into an amorphous form).

Porating

A method of producing a cartilage product of the present invention(“method of manufacture”) optionally comprises a step of porating acartilage sample. The poration can be conducted in any manner thatincreases the flexibility of the cartilage and provides a plurality ofpassageways through which cells and factors can migrate.

In one embodiment, the cartilage is porated using laser poration ormechanical proration.

In one embodiment, the cartilage is porated using mechanical poration.Optionally, the mechanical poration is provided by drilling, punching,hydraulic poration (e.g. high pressure fluid drilling), or combinationsthereof. Optionally, the cartilage is porated using a single punch or amulti-punch device.

In one embodiment, the cartilage sample is porated to an extent thatincreases flexibility of the cartilage sample. Optionally, the cartilagesample is porated to provide pores having a diameter of about 0.25 mm toabout 2 mm (e.g. about 0.25 mm to about 1.5 mm or about 0.5 mm to about1.5 mm) in diameter. Optionally, the cartilage sample is porated toprovide about 10 to about 400 pores per cm² such about 10 to about 100pores per cm² or about 20 to about 60 pores per cm² (e.g. about 36 poresper cm²). Optionally, the cartilage sample comprises a layer ofcartilage and the pores pass through the majority (e.g. entirety) of thelayer of cartilage.

In one embodiment, the cartilage sample is porated to an extent of about10 mm² to about 50 mm² of porated surface area per cm² of surface areaof the cartilage sample. For example, a collagen matrix comprisingcylindrical pores with a 1 mm diameter at a pore density of 36 pores percm² would comprise about 28 mm² of porated surface area per cm² [(0.5 mmpore radius)×(π)×(36 pores/cm²)].

In one embodiment, porating the cartilage sample comprises chilling(e.g. continually or periodically) the sample, e.g. using chilledsolvent and/or a cold room or a cold plate. In one embodiment, poratingthe cartilage sample comprises porating the cartilage sample withoutgenerating a substantial amount of heat, e.g. using a low speed drill ora tissue punch.

Digestion

A cartilage product of the invention is optionally produced by partiallydigesting a cartilage sample, e.g. using a digestive enzyme such as aproteinase or proteoglycan digesting enzyme.

According to manufacturing methods of the present invention, a step ofpartial digestion modifies the ECM of the cartilage sample and can beperformed in a manner that provides one or more of the followingtechnical features:

-   -   a loose ECM that releases and allows migration of cellular        factors and viable cells.    -   a natural ECM retaining viable native cells    -   preservation of physiologic interactions between cells and the        ECM    -   a clean cartilage product devoid of debris    -   a cartilage product comprising ECM fibrils that substantially        retain the packing density of natural cartilage    -   a cartilage product with greater flexibility    -   removes macrophages and reduces immunogenicity

In one embodiment, the step of partial digestion is performed in amanner that loosens the ECM, e.g. cleaving peptide bonds withincollagen. A loose ECM releases and allows migration of cellular factorsand viable cells. For example, bio- (e.g. chondrogenic) factors canleach out into the surrounding micro environment upon administration toa subject. With the teachings provided herein, one skilled in the artcan now tailor digestion to provide such a technical feature.

In one embodiment, the step of partial digestion is performed in amanner that retains a substantial amount of viable native cells, forexample, by limiting digestion to a cell-sparing amount. With theteachings provided herein, one skilled in the art can now tailordigestion to provide such a technical feature.

In one embodiment, the step of partial digestion is performed in amanner that preserves normal interactions between cells and the ECM. Forexample, the ECM and/or cellular factors therein activate chondrocytes,i.e. induce a shift from the G0 phase to the G1 phase, and also induceMSCs to infiltrate and differentiate into chondrocytes. Without beingbound by theory, the inventors believe that these functions enhancetherapeutic efficacy.

In one embodiment, the step of partial digestion is performed in amanner that cleans the cartilage sample of debris. This microscopicand/or macroscopic debris (e.g. ECM fragment) is present in even greateramounts upon poration of a cartilage sample, and can trigger pain andother adverse responses when administered to a subject if the cartilageproduct of the present technology is not cleansed of debris.

In one embodiment, the step of partial digestion is performed in amanner that provides fragmented ECM fibrils that substantially retainthe packing density of natural cartilage. For example, partial digestioncan be limited to an amount that does not destroy the structuralintegrity of the cartilage sample. Such a technical feature provides acartilage product having mechanical properties of native cartilage, forexample, to provide a long lasting, weight-bearing cartilage graft.

In one embodiment, the step of partial digestion is performed in amanner that imparts flexibility to the cartilage product.

In one embodiment, the cartilage sample is digested using acollagen-digesting enzyme (e.g. collagenase or cathepsin) or aproteoglycan-digesting enzyme (e.g. hyaluronidase, aggrecanase, orpapain). Optionally, the cartilage sample is digested in a manner thatretains cell-matrix interaction. For example, trypsin digestion istypically performed with a chelator such as EDTA to sequester magnesiumand calcium, which otherwise inhibit the action of trypsin. Suchchelators can dissociate cells from the matrix. Indeed, trypsin itselfcan cut matrix proteins to which cells adhere or attach. Accordingly,one embodiment of the invention contemplates the use of non-dissociativedigests that can partially digest a cartilage sample in a manner thatretains native cells such as chondrocytes.

In one embodiment, the step of partial digestion comprises digestingcollagen II in a cartilage sample (e.g. articular cartilage). Digestionenzymes which are useful in the partial digestion of type II collagenmatrices include: collagenase (e.g. Type II, any of collagenase I-IV,and bacterial collagenase), other endopeptidases (e.g. trypsin, papain,pepsin), and exopeptidases (e.g. carboxypeptidase).

In one embodiment, partial digestion comprises non-enzymatic digestion,for example, steam-based, acid-based, or fenestration-based digestion.Optionally, the non-enzymatic digestion is mechanical digestion, e.g.partial mincing or fenestration-based digestion.

In some embodiments, the cartilage products of the present invention arenot digested. In other embodiments, the cartilage products of thepresent invention are partially digested by digestive means includingenzymatic (e.g. collegenase, pronase, proteinase K, etc. treatment),biochemical (e.g. papain), thermal (e.g. increased heat), chemical(keratin sulfate, tosyllysylchloromethane), mechanical (perforated), anyother means of digestion known by those of skill in the art, andcombinations of any two or more of the foregoing.

Cryopreservation

A cartilage product of the present invention may be used fresh or may bepreserved for a period of time. Optionally, the cartilage product issubjected to a freeze-thaw cycle, i.e. cryopreserved and then thawed.

In one embodiment, a cartilage product is cryopreserved. A cartilageproduct may be cryopreserved by incubation at freezing temperatures(e.g. at −80° C.±5° C.) in a cryopreservation medium.

In one embodiment, cryopreservation can comprise a controlled method offreezing, i.e. wherein the cartilage product is held at one or moretemperatures intermediate of room temperature and −80° C.Cryopreservation can comprise, for example, incubating the cartilageproduct at 4° C. for 30 min to 24 hrs (e.g. about 30 to about 90 min),then incubating the cartilage product at about −20° C. to about −40° C.(e.g. about −30° C.) for about 20 min to about 12 hrs (e.g. about 20 toabout 60 min) and then incubating at −80° C. until use for example, byreducing the temperature at a rate of about 4° C./min to about −80°C./min. Alternatively, the cartilage product can be rapidly frozen at−80° C. or snap frozen in liquid nitrogen.

The cartilage product may then be thawed for use. Optionally, thecartilage product is cryopreserved in a manner such that cell viabilityis retained surprisingly well after a freeze-thaw cycle.

In one embodiment, cryopreservation comprises storage in acryopreservation medium comprising one or more cell-permeatingcryopreservatives, one or more non cell-permeating cryopreservatives, ora combination thereof. Optionally, the cryopreservation medium comprisesone or more cell-permeating cryopreservatives selected from DMSO, aglycerol, a glycol, a propylene glycol, an ethylene glycol, or acombination thereof. Optionally, the cryopreservation medium comprisesone or more non cell-permeating cryopreservatives selected frompolyvinylpyrrolidone, a hydroxyethyl starch, a polysacharide, amonosaccharides, a sugar alcohol, an alginate, a trehalose, a raffinose,a dextran, or a combination thereof. Other examples of usefulcryopreservatives are described in “Cryopreservation” (BioFiles Volume 5Number 4-Sigma-Aldrich® datasheet).

In one embodiment, the cryopreservation medium comprises acell-permeating cryopreservative, wherein the majority of thecell-permeating cryopreservative is DMSO.

In one embodiment, the cryopreservation medium comprises DMSO, e.g.about 1% to about 50% DMSO by volume (e.g. about 10%).

In one embodiment, the cryopreservation medium comprises additionalcomponents such as albumin (e.g. HSA or BSA), an electrolyte solution(e.g. Plasma-Lyte), or a combination thereof.

In one embodiment, the cryopreservation medium comprises 1% to about 20%albumin by weight and about 1% to about 50% cryopreservative by volume(e.g. about 10%). Optionally, the cryopreservative comprises DMSO (e.g.in a majority amount).

Antiseptic Treatment

A cartilage product of the present invention is optionally treated withone or more antiseptic solutions to reduce bioburden. Optionally, thecartilage product is treated with (e.g. incubated in) an antibiotic.Optionally, the cartilage product is treated (e.g. wiped down) withpovidone-iodine.

In some embodiments, the cartilage product is treated with anantibiotic, where the antibiotic is gentamicin sulfate (AbraxisPharmaceutical Products, Schaumburg, Ill.), vancomycin HCl (HospiraInc., Lake Forest, Ill.), and/or amphotericin B (Sigma Aldrich, St.Louis Mo.).

Optionally, the cartilage product is treated with a fungicidal solution.

Screening for Cells, Viability, and Chondrogenic Factors

In one embodiment, a cartilage product is screened for chondrocytes,cell viability, and one or more structural or functional components suchas bioactive factors and other ECM components (e.g. chondrogenicfactors).

Through the insight of the inventors, it has been discovered thatcertain components correlate with therapeutic efficacy.

In one embodiment, the components screened for include one or more ofthe bioactive factors listed in Table 4, the presence of viablechondrocytes, or a combination thereof.

Miscellaneous

In one embodiment, a method of manufacturing a cartilage product of thepresent invention comprises treating the cartilage product with one ormore solutions. Optionally, the pH of the one or more solutions rangesfrom 5-10. Optionally, treatment solutions comprise one or more of:saline, PBS, Plasmalyte, and water.

Methods of Use

In one embodiment, a cartilage product of the present invention is usedto treat an injured tissue in a subject. The injured tissue can be anyinjured tissue. A method of treatment may be provided, for example, byadministering to a subject in need thereof, a cartilage product of thepresent invention.

In one embodiment, the injured tissue is cartilage. Optionally, theinjured tissue is articular cartilage. Optionally, the method comprisesremoving injured cartilage at and administering the cartilage product tothe site at which the injured cartilage was removed. Optionally, thestep of removing comprises removing a plug comprising the injured tissueand the cartilage product is cut or shaped (or both) to fit the voidleft by removing the plug (e.g. 2 cm diameter plug is removed andreplaced with a 2 cm diameter cartilage product in the shape of saidplug).

In one embodiment, the injured tissue is articular cartilage and themethod further comprises microfracturing the injured tissue site, e.g.removing damaged articular cartilage followed by physically insultingthe underlying subchondral bone to exposed bone marrow and createbleeding. Optionally, the cartilage product is placed on the subchondralbone after microfracturing.

Microfracturing is a technique that can stimulate healing by forming ablood clot, thereby introducing inflammatory cytokines, growth factorsand MSCs. Microfracturing alone results in the formation offibrocartilage comprising collagen type I and has poor biomechanicalperformance and abnormal bone growth resulting in osteoarthritis.Surprisingly, through the insight of the inventors, it has beendiscovered that the present cartilage products overcome thesedeficiencies by providing a type II collagen matrix with viablechondrocytes to promote chondrogenesis of the MSCs introduced bymicrofracturing.

In one embodiment, a cartilage product of the present invention isadministered arthroscopically. Surprisingly, a flexible cartilageproduct of the invention can be easily administered arthroscopically(i.e. is flexible such that it can be administered through anarthroscope cannula) and adapts to contours at the site ofadministration, e.g. contours of chondral surfaces.

In one embodiment, a cartilage product is fixed at the site ofadministration. Optionally, the cartilage product if fixed by anadhesive (e.g. fibrin glue) or by a mechanical device (e.g. a pin suchas a bioresorbable pin).

Surprisingly, through the insight of the inventors, cartilage productsof the present invention provide greater healing and can be used totreat injuries of larger sizes.

Surprisingly, through the insight of the inventors, cartilage productsprovide a collagen matrix with native mechanical and functionalproperties that is efficiently integrated into the cartilage at thetarget site. Without being bound by theory, it is believed that thesuperior healing capacity of cartilage products taught herein is due, inpart to complex interactions between the donor matrix (cartilageproduct), donor cells, recipient (treated subject) cells, and recipientmatrix. This is far superior to prior therapies such as autologouschondrocyte implantation which are typically only palliative.

EXAMPLES Example 1 Isolation of Femoral Condyles and Tibial Plateau

A human knee joint was obtained, as depicted in FIG. 1.

The outer surfaces of the knee joint were cleaned with iodine (10%povidione-iodine solution, Purdue, “Betadine”) without contacting thecartilage with iodine. The knee joint was dissected to separate thefemur, tibia and fibula without damaging the cartilage surfaces. Softtissue (adipose, muscle, fascia, ligaments and tendons) were removed toexpose the articular cartilage surfaces on tibial plateau and femoralcondyles.

The portions containing the articular cartilage (tibial plateau and thecondyles of the femur) were chilled by placing in chilled saline (0.9%Sodium Chloride irrigation solution, USP) on a cold plate.

Example 2 Isolating Cartilage Plugs

Femur condoyles and tibial plateau were obtained as detailed inExample 1. Osteochondral plugs having diameters of about 1 cm or about 2cm were obtained from the femur condoyles and tibial plateau. Duringisolation of the plugs, the condoyles and tibial plateau were kept moistand chilled by periodic immersion in chilled saline or wiped with a wipesoaked in chilled saline. The isolated plugs were then chilled byplacement in chilled saline.

The osteochondral plugs were obtained using a tissue punch whileavoiding any areas of damaged cartilage. Specifically, tissue puncheswith diameters of 1 cm or 2 cm were used to remove whole plugs ofcartilage and underlying bone from the articular surface.

Example 3 Isolating a Cartilage Sample from Subchondral Bone andCalcified Cartilage

Osteochondral plugs were obtained as detailed in Example 2. Thesubchondral bone and calcified cartilage was removed from theosteochondral plugs to provide cartilage samples in the form ofcartilage disks. During this process, the cartilage was chilledperiodically with chilled saline to prevent overheating.

Specifically, each osteochondral plug was held securely and thesubchondral bone layer was cut (removed) using a sagittal saw with abent angle blade from the layer of cartilage. Once the subchondral bonewas removed, any remaining bone and calcified cartilage was shaved fromthe underside of the cartilage discs. To prevent overheating, the tissuewas frequently immersed in chilled saline throughout the sawing andshaving process. This process was repeated for each of the cartilagedisks.

Example 4 Sizing of Cartilage Samples

Isolated cartilage samples in the form of cartilage disks were obtainedas detailed in Example 3, and then sized to increase their flexibility.The thickness of cartilage samples was measured using a caliper or discthickness gauge. Disks that were thicker than about 1.5 mm were trimmeddown to about 1.5 mm. Disks thinner than about 1 mm were discarded.

Example 5 Porating a Cartilage Sample

Cartilage samples (cartilage disks) were obtained and sized as detailedin Example 4 and then porated to provide a cartilage layer (disk) havingpores of about 1 mm in diameter with a pore density of about 36pores/cm² as depicted in FIG. 15.

Specifically, a pore pattern (a perforated stainless steel screen) wasplaced over the cartilage sample and a 1 mm biopsy punch was used topunch out the pores (holes) of cartilage through the pattern. Theporated cartilage sample was then chilled by immersion in chilledsaline.

Example 6 Partial Digestion of a Cartilage Sample

Cartilage samples (cartilage disks) were obtained as detailed in Example5 and then digested with Type II collagenase.

Specifically, the collagenase was formulated in DMEM (200 units/ml ofcollagenase type II, Sigma). The porated cartilage samples wereincubated in the collagenase suspension for 30±2 minutes at 37° C.±2° C.The collagenase solution was removed and the disks were rinsed withchilled saline.

Example 7 Antibiotic Treatment of a Cartilage Product

Cartilage samples (cartilage disks) were obtained as detailed in Example7 and then incubated antibiotic solution containing of gentamicinsulfate (50 μg/mL; Abraxis Pharmaceutical Products, Schaumburg, Ill.),vancomycin HCl (50 μg/mL; Hospira Inc., Lake Forest, Ill.), andamphotericin B (2.5 μg/mL; Sigma Aldrich, St. Louis, Mo.) in DMEM at 37°C.±2° C. for 18 hrs to 48 hr. Following the incubation, the antibioticsolution was removed and the disks were rinsed in chilled saline.

Example 8 Cryopreservation

Cartilage products were obtained as detailed in Example 8 andcryopreserved in a cryopreservation solution.

The cryopreservation solution contained 10% dimethyl sulfoxide (DMSO)(Bioniche Teo. Inverin Co) and 5% human serum albumin (HSA; Baxter) inPlasmaLyte-A (Baxter Healthcare Corp.).

For each cartilage product, a vial was filled with about 7 ml of thecryopreservation solution and a cartilage product was transferred intothe vial using forceps. The cartilage product was wiped to remove anyresidual liquid (e.g. saline solution) prior to placement in thecryopreservation solution. A stopper was placed in the vial containingthe cryopreservation solution and the cartilage product.

The vial was sealed after capping, crimping, and bagging the vial, andthen cryopreserved at about −80° C. by placing the vial in an automatedfreezer. The freezer was programmed to reduce the temperature in agradual and step-wise manner using the following temperature program:

Step 1 reduce temperature to 4° C. at 4.0° C./m

Step 2 hold temperature for 60 m at 4° C.

Step 3 reduce temperature to −30° C. at 1.0° C./m

Step 4 hold temperature at −30° C. Hold for 30 m

Step 5 reduce temperature to −80° C. at 4.0° C./m

Step 6 hold temperature at −80° C.

Example 9 Cartilage Product

Cartilage products were produced by isolating, porating, digesting, andcryopreserving a cartilage sample using the method detailed in Example 1through Example 8. The cartilage products have a natural structuralorganization and promote proper articular cartilage repair.Surprisingly, therapeutically active cartilage products have thefollowing technical features:

-   -   contain viable native chondrocytes having a capacity for        chondrogenesis    -   contain bioactive factors    -   are non-immunogenic    -   provide a flexible repair matrix having a natural structural        organization

Example 10 Viable Chondrocytes After Cryopreservation Using VariousFreezing Methods

Cartilage products from Example 9 were analyzed for viable chondrocytesafter cryopreservation.

Various freezing methods were investigated to determine their effect oncell viability. Cartilage products were formulated in a cryopreservationmedium comprising 10% Dimethyl Sulfuric oxide (DMSO) and 5% Human SerumAlbumin (HSA) in plasmalyte-A (sodium chloride, sodium gluconate, sodiumacetate, potassium chloride and magnesium chloride).

In a first set of experiments, different cryopreservation methods weretested to determine the effect on preservation viable cells within thecartilage product.

Method 1: Hold the cartilage product at 4° C. to allow time forequilibration (i.e. penetration of cryosolution into the tissue),followed by placing the product in a styrofoam box and freezing in a−80° C. freezer which results in a uniform cooling rate of about −0.5°C./min.

Method 2, Step:

-   -   1. Reduce the temperature in a step-wise manner:    -   2. reduce temperature to 4° C. at 4.0° C./m    -   3. hold temperature for 60 m at 4° C. for equilibration    -   4. reduce temperature to −30° C. at 1.0° C./m    -   5. hold temperature at −30° C. Hold for 30 m    -   6. reduce temperature to −80° C. at 4.0° C./m    -   7. hold temperature at −80° C.

To demonstrate the presence of viable cells, the cartilage products werestained using a LIVE/DEAD® Viability/Cytotoxicity kit (Molecular ProbesInc., Eugene, Oreg.) to qualitatively assess cell viability. Stainingwas performed as per the manufacturer's protocol. Thin portions ofcartilage products (e.g. approximately 0.5 cm×0.5 cm×0.02 cm) werethawed in a 37° C. water bath and used for analysis. Evaluation ofstained tissue was performed using a fluorescence microscope. An intenseuniform green fluorescence indicated the presence of live cells, and abright red fluorescence indicated the presence of dead cells.

As depicted in FIG. 3, cartilage products cryopreserved by the stepwisemethod (Method 2; FIG. 3 b) comprise enhanced levels of viablechondrocytes relative cartilage products cryopreserved using a methodcomprising a single equilibration step followed by gradual cooling(Method 1; FIG. 3 a). Chondrocytes are the predominant cell type presentwithin articular cartilage and are integral in maintaining cartilagematrix homeostasis. Additionally, chondrocytes express factors thatpromote chondrogenesis and cartilage repair. It is quite surprising thatviable chondrocytes remain following cryopreservation as severalattempts have been cited in the literature to cryopreserve cartilagewith little success in preserving viable cells once thawed (e.g. Acostaet al, 2007. Clin Orthop Relat Res; 460:234-9).

In a second set of experiments, the duration of equilibration was testedfor its effect on the level of viable chondrocytes in thawed cartilageproducts. Specifically, cell viability was accessed aftercryopreservation using method 2 (FIG. 4 a) and compared to methods ofcryopreservation that substituted a 2 hour equilibration step (FIG. 4 b)or 4 hour equilibration step (FIG. 4 c) for the 1 hour equilibrationstep at 4° C. Surprisingly, as depicted in FIG. 4, a 1 hourequilibration step provided comparable (or better) cell viability tothat of 2 hour and 4 hour equilibrations.

Example 11 Viable Chondrocytes after Cryopreservation in VariousCryopreservation Media

Cartilage products from Example 9 were analyzed for viable chondrocytesafter cryopreservation. Various cryopreservation media were investigatedto determine their effect on cell viability.

In a first experiment, cartilage products were formulated in acryopreservation medium containing either 10% or 20% DMSO in 5% HSA inplasmalyte A. LIVE/DEAD® staining was performed on thawed cartilageproducts of the final cartilage product in the manner detailed inExample 10. The results are depicted in FIG. 5. There was no qualitativedifference in cell viability between 10% (FIG. 5A) and 20% DMSO (FIG.5B). Accordingly, the cartilage product can be formulated in reducedconcentrations of cryopreservation medium to provide products withviable chondrocytes upon thawing.

In a second experiment, cartilage products were formulated in acryopreservation medium containing either 0% or 5% HSA and 10% DMSO in5% HSA in plasmalyte A. LIVE/DEAD® staining was performed on thawedcartilage products of the final cartilage product in the manner detailedin Example 10. The results are depicted in FIG. 6. In this study, noremarkable difference was observed in cell viability between 0% (FIG.6A) and 5% HSA (FIG. 6B). However, through inventor insight regardingthe effect of HSA on long term stability of cryopreserved tissues, HSAis optionally included.

In a third experiment, cartilage products were formulated in acryopreservation medium containing 10% DMSO+5% HSA solution in eitherplasmalyte-A or normal saline. LIVE/DEAD® staining was performed onthawed cartilage products of the final cartilage product in the mannerdetailed in Example 10. The results are depicted in FIG. 7. The resultsdid not demonstrate a remarkable difference in cell viability betweenplasmalyte-A (FIG. 7A) and normal saline (FIG. 7B). However, throughinventor insight, plasmalyte-A is optionally included because it containsalts and minerals (e.g. sodium chloride, sodium gluconate, sodiumacetate, potassium chloride and magnesium chloride) that may bebeneficial for long term stability of the cartilage product duringcryogenic storage. A useful cryosolution for cartilage products of thepresent invention include 10% DMSO+5% HSA in plasmalyte-A.

These results indicate that the cartilage products taught herein containviable chondrocytes after a freeze/thaw cycle when formulated in variouscryopreservation media.

Example 12 Sustained Viability of Cells after Thawing

Cartilage products from Example 9 were analyzed for viable chondrocytesafter cryopreservation. Cell viability was determined at various timepoints after thawing cartilage products.

Cartilage products were formulated in a cryopreservation mediumcontaining either 10% DMSO in 5% HSA in plasmalyte A. Aftercryopreservation and thawing, cartilage products were cultured inDMEM+1% HSA+antibiotic/antimycotic for up to 14 days. At 0 days (FIG.8A), 7 days (FIG. 8B) and 14 days (FIG. 8C) of culturing, cartilageproducts were evaluated for LIVE/DEAD® staining in the manner detailedin Example 10. As depicted in FIG. 8, chondrocytes were viable duringthe 14 day culture period after a freeze/thaw cycle. Thus, cartilageproducts of the present invention provide a cellular component thatcontributes to therapeutic efficacy.

Example 13 Quantitative Evaluation of Cell Viability

Cartilage products from Example 9 were analyzed for cell number and cellviability after cryopreservation.

Cryopreserved cartilage products were thawed and thin sections werestained with LIVE/DEAD® staining as detailed in Example 10. Viable anddead chondrocytes were visualized under a 10× magnification lens andcounted as indicated by either green or red fluorescence, respectively,within a 0.38 mm² field. Three random sections were analyzed from fourseparate donors. As detailed in Table 1, the average number of viablecells was 64,989 cells/cm² with cell viability of 70.5%. This dataindicates that cartilage products of the present invention can have 70%viability.

TABLE 1 Cell Viability Live cells/cm² Dead cells/cm² Viability Donor 50Section 1 46,579 8,421 85.0% Section 2 38,947 10,000 79.6% Average42,763 9,211 82.3% Donor 53 Section 1 95,789 13,421 87.7% Section 261,316 17,632 77.7% Section 3 67,895 53,158 56.1% Average 75,000 28,07072.8% Donor 54 Section 1 57,632 5,789 90.9% Section 2 66,316 11,08185.7% Section 3 76,579 9,211 89.3% Average 66,842 8,694 89.3% Donor 55Section 1 83,158 82,368 50.2% Section 2 63,947 18,684 77.4% Section 378,947 87,895 47.3% Average 75,351 62,982 54.5% Total Average 64,98927,239 70.5%

Example 14 Flexible Cartilage Product

In one embodiment, cartilage products of the present invention exhibitenhanced flexibility allowing them to be administered arthroscopically.Surprisingly, by poration and optional digestion, a cartilage productcan be made flexible enough to be threaded through an arthroscopiccannula; this is in contrast native articular cartilage which isnormally hard with very little ability to flex without breakage.

Various pore sizes and pore densities were evaluated to determine theireffect on flexibility and capacity for use in arthroscopy. Cartilageproducts were produced using the method detailed in Example 4 andprocessed further by poration alone or poration and digestions. Twodifferent pore sizes were tested, 0.6 mm and 0.9 mm diameter pores.Three different pore densities were tested: 12, 25 and 50 pores/cm². Inaddition, a 30 minute collagenase digestion was also tested to evaluatethe effect of digestion on the cartilage product. Various combinationsof treatment conditions (treatments A-L) were evaluated, as detailed inTable 2. Each of the cartilage products produced by treatments A-L is anexemplary cartilage product of the present invention.

Specifically, each of treatment conditions A-L was labeled with acorresponding letter and 6 blinded evaluators were asked to rate thecartilage product for flexibility on a scale from 1-5 (1=most flexibleand 5=hardest and least flexible). The results are depicted in Table 3.The results indicate that a larger pore size (0.9 mm diameter) andgreater pore frequency (50 pores/cm²) yielded the most flexiblecartilage product. In this experiment, collagenase treatment did notdemonstrate a remarkable difference in flexibility. However, in otherexperiments (data not shown), users observed much more marked change inflexibility due to collagenase treatment.

TABLE 2 Treatment Conditions Treatment Description A 0.6 mm pores, 12pores/cm² B 0.6 mm pores, 25 pores/cm² C 0.6 mm pores, 50 pores/cm² D0.9 mm pores, 12 pores/cm² E 0.9 mm pores, 25 pores/cm² F 0.9 mm pores,50 pores/cm² G 0.6 mm pores, 12 pores/cm² + collagenase treatment H 0.6mm pores, 25 pores/cm² + collagenase treatment I 0.6 mm pores, 50pores/cm² + collagenase treatment J 0.9 mm pores, 12 pores/cm² +collagenase treatment K 0.9 mm pores, 25 pores/cm² + collagenasetreatment L 0.9 mm pores, 50 pores/cm² + collagenase treatment

TABLE 3 Flexibility of Cartilage Products After Various TreatmentConditions Eval Eval Eval Condition Eval #1 #2 Eval #3 #4 Eval #5 #6Average A 5 5 4 5 5 5 4.8 B 5 4 4 5 5 5 4.7 C 4 4 4 3 3 3 3.5 D 5 4 3 44 2 3.7 E 4 4 3 3 3 1 3.0 F 3 3 2 2 2 1 2.2 G 5 5 5 5 5 5 5.0 H 5 5 5 55 5 5.0 I 4 4 5 4 4 3 4.0 J 5 4 4 4 3 2 3.7 K 4 4 3 3 4 1 3.2 L 3 3 2 22 1 2.2

Example 15 Non-immunogenicity of Cartilage Products

Cartilage products from Example 9 were analyzed for immunogenicity.Specifically, secretion of TNF-α by cartilage products in response tolipopolysaccharide (LPS) was used to determine immunogenicity. Thesecretion of TNF-α of cryopreserved cartilage products of the presentinvention was compared to that of raw (fresh) cartilage products

Pieces (0.785 cm²) of cartilage products (raw vs. cryopreserved) wereplaced in tissue culture medium and exposed to bacterial LPS (1 μg/mL,Sigma) for 20-24 hr. After 24 hours, tissue culture media were collectedand tested for the presence of TNF-α using a TNF-α ELISA kit (R&DSystems) according to manufacturer's protocol. Human hPBMCs, known tocontain monocytes that secrete high levels of TNF-α upon LPSstimulation, were used as a positive control in the assay. hPBMCs andcartilage products without LPS were also included as controls in theanalysis.

The results are depicted in FIG. 9. Non-cryopreserved cartilage products(“raw product”) provided substantial levels of TNF-α in response to LPSwhile cryopreserved cartilage products (“final product”) did not providesubstantial levels of TNF-α in response to LPS, indicating that themanufacturing process eliminates immunogenicity of cartilage samples.Without being bound by theory, the inventors believe that viablefunctional macrophages are the source of immunogenicity in theunprocessed cartilage.

Surprisingly, these results indicate that cartilage products can beselectively depleted of macrophages to reduce immunogenicity ofallogeneic implants.

Example 16 Bioactive Factors in Cartilage Products

Cartilage products from Example 9 were analyzed for the presence ofbioactive factors. Specifically, Enzyme-Linked Immunosorbent Assays(ELISAs) were used to analyze tissue extracts and factors released incultured supernatants of the cartilage products.

For the tissue extract assay, cryopreserved cartilage products ofExample 9 were thawed in a 37° C. water bath and removed from thecryopreservation medium followed by a PBS rinse. Each product was thenfinely minced and snap frozen in a homogenization tube in a liquidnitrogen bath. One pre-cooled 5 mm steel bead was added to each tube andhomogenized using a Qiagen Tissue Lyser according to the manufacture'srecommendations in 1 ml homogenization media. Homogenates were then spundown at 16000 g for 10 minutes using a microcentrifuge. Supernatantswere collected and stored at −80° C. until analyzed by ELISA for proteinexpression. The supernatant volume was approximately equal to that ofthe initial volume of homogenization media (1 ml).

For the factor release assay, cryopreserved cartilage products werethawed in a 37° C. water bath and removed from the cryopreservationmedium followed by a PBS rinse. Each cartilage product was plated onto awell of a 12-well dish and 2 ml of growth media (DMEM+1% HSA+antibiotic/antimycotic) was added and incubated at 37° C. for up to 14days. After incubation, tissue and culture media were transferred to a15 ml conical tube and centrifuged at 2000 rpm for 5 min. Culturesupernatant was collected analyzed by ELISA for protein expression. Thesupernatant volume was approximately equal to that of the initial volumeof growth media (2 ml).

Table 4 lists examples of chondrogenic factors detected in the tissueextract and factor release assays. Each expression value is provided interms of amount of factor per supernatant volume per superior surfacearea (identified in FIG. 15) of the cartilage product (pg/ml/cm²) andamount of factor per superior surface area of the cartilage product(pg/cm²).

TABLE 4 Chondrogenic Factors Range of factors released in culturesupernatants Range of expression (adjusted per cm² in tissue lysates oftissue) Factor (pg/cm²) (pg/ml) TGF-β1  10.8-627.8 2616.6-17818  TGF-β2TBD 133-623 TGF-β3  3.98-112.1 TBD BMP-2 TBD TBD BMP-7 3.33-23.3 TBDbFGF 168.8-365  TBD IGF-1 111-779  14-2842 ECM (Collagen type II, TBDTBD Hyaluronan, Aggrecan)

Without being bound by theory, the inventors believe that bioactivefactors (e.g. growth factor proteins) that mediate extracellular matrixproduction and promote chondrogenesis are important to efficientcartilage repair as facilitated by cartilage products of the presenttechnology.

Surprisingly, these results indicate that the cartilage productcomprises a variety of chondrogenic factors that facilitate therapeuticvalue in articular cartilage repair.

Example 17 Sustained Release of Proteins from Cartilage Products

Cartilage products of the present invention can release factors into themicroenvironment by cells or tissues to enhance their functionalactivity.

To measure the amount or proteins released, cartilage products ofExample 9 were cultured in culture media between 7-21 days andsupernatants were collected and key proteins were quantified by ELISA.The results are depicted in FIG. 10, which indicate that the cartilageproducts produce and release TGF-β1 and TGF-bβ into the supernatant forat least 21 days. These data suggest that the cartilage product has theability to produce and sustain chondrogenic growth factors levels overtime due to the presence of viable chondrocytes and dense ECM

Example 18 Factor Release from Porated Cartilage Products

The effect of poration versus intact cartilage products on protein wasinvestigated. Cartilage products were generated as detailed in Example 9except that the poration parameters were modified. Half the productswere porated between 36-50 pores/cm² while the rest were kept intactwith no poration. The amount of TGF-β1 was measured by ELISA fromsupernatants of both conditions of cartilage products cultured for 7days. The results are depicted in FIG. 11, which indicate that theamount of TGF-β1 released from porated cartilage implants is greaterthan intact cartilage products. These data indicate that not only do theporations within the product contribute to the flexibility of theproduct but poration also supports greater release of chondrogenicfactors. Without being bound by theory, the inventors speculate that theenhanced release is due to the increased surface area created by thepores.

Example 19 Factor Release from Digested Cartilage Products

The effect of digestion of cartilage products on protein release wasinvestigated. Cartilage products were generated as detailed in Example 9except that the digestion parameters were modified. Half the productsdid not undergo the 30 minute collagenase digestion prior tocryopreservation. The amount of TGF-β1 released was measured by ELISAfrom supernatants of both conditions of products cultured for 14 days.The results are depicted in FIG. 12 which demonstrates that the amountof TGF-β1 released from collagenase digested cartilage products isgreater than non-digested cartilage products. These data indicate thatnot only does collagenase digestion contribute to the flexibility andcleanliness of the product but digestion also supports greater releaseof beneficial proteins to the microenvironment.

Example 20 TGF-β Factor Release from Cryopreserved Cartilage ProductsContaining Live Cells

The effect of cryopreservation on protein release was investigated.Cartilage products were generated as detailed in Example 9 (i.e.cryopreserved). Next, some cartilage products underwent an additionalthree freeze thaws in H₂O to kill all the cells within the product(“devitalization”). As a final step, all cartilage products were thawedand cultured in separate wells in growth media for 21 days.

The amount of TGF-β1 spontaneously released into the media was measuredby ELISA from supernatants of both conditions of cartilage productscultured for the 21 days. The results are depicted in FIG. 13, whichdemonstrates that the amount of factor (TGF-β1) released fromcryopreserved cartilage products containing live cells is greater thandevitalized cartilage products all throughout the 21 day culture. Thesedata indicate that cryopreserved cartilage products contain viable cellsthat continue to produce and contribute beneficial factors such asTGF-β1 to the microenvironment as compared to cartilage without livingcells.

Example 21 Cell Viability after Povidone-Iodine Treatment of CartilageProducts

Efforts to optimize the aseptic processing of donor tissue are importantfor therapeutic use (and, e.g., in order to comply with the Food & DrugAdministration (FDA) and tissue bank regulations regarding tissueproduct safety). To minimize incoming bioburden carried by the donortissue, cartilage products were treated with an overnight antibioticincubation prior to cryopreservation, as detailed in Example 7. Tofurther decrease bioburden of the cartilage product, povidone-iodinetreatment was tested to observe any changes in cell viability or proteinexpression. Povidone-iodine is a potent antiseptic widely used to in theclinic to cleanse and decontaminate surgical surfaces.

Briefly, cartilage products were produced as detailed in Example 9,however, prior to overnight antibiotic incubation, cartilage productswere submerged in a povidone-iodine bath for 1 sec and then immediatelywashed in saline 3 times. Cartilage products then followed the normalantibiotic incubation and cryopreservation process. To assess the effectof povidone-iodine treatment, cell viability (LIVE/DEAD® staining) wasanalyzed as detailed in Example 10. The results are depicted in FIG. 14.Three concentrations of povidone-iodine were tested, 10% povidone-iodine(FIG. 14A), 5% povidone-iodine (FIG. 14B), 1% povidone-iodine (FIG.14C), and 0% povidone iodine as a control (FIG. 14D). The LIVE/DEAD®staining of thawed cartilage products revealed that with increasingconcentrations of povidone-iodine, cell viability also decreasedcompared to the control untreated cartilage product.

Example 22 Treatment of Chondral Defects in Goats with CartilageProducts

In one embodiment, the efficacy of a cartilage product is tested in ananimal model, e.g. an animal model of focal chondral defects.

Briefly, focal chondral defects are induced into stifle joints of a goatand then treated with microfracture alone or microfracture withcartilage products (as produced in Example 9). At 3, 6 and 12 months,joints are collected and repair tissue is analyzed for volume of defectfilling as well as collagen type II staining indicating formation ofarticular cartilage repair tissue. In addition, goats can be evaluatedfor safety of the product by monitoring for inflammation or generaldiscomfort of the animal throughout the duration of the study.

Cartilage products of the present technology substantially increasecartilage repair according to at least one or more of the followingnon-limiting criteria.

-   -   Safety    -   Gross morphology    -   Quality of repair tissue relative to native surrounding tissue    -   Integration of repair tissue    -   Histological evaluation    -   Extracellular matrix staining    -   Defect volume filling    -   Mechanical evaluation    -   Indentation testing of repair tissue    -   Repair tissue evaluation via O'Driscoll grading system

Example 23 Chondrogenesis of Viable Cells within Cartilage Products

This study demonstrates that the viable cells within the cartilageproduct are functional and have the potential to lay down healthy ECMthat will contribute to proper cartilage repair.

Chondrocytes are isolated and expanded from cartilage products of thepresent invention. Prolonged in vitro culturing of chondrocytes resultsin dedifferentiation of some cells (e.g. to a more primitivefibroblastic lineage). Next, these cells are placed in a differentiationmedium (e.g. containing growth factors). Over time, these cellsdemonstrate chondrogenesis in vitro.

Example 24 Gene Expression of Chondrocytes within Cartilage Products

Cartilage products are generated as detailed in Example 9 (except, withand without cryopreservation). Chondrocytes within the cartilage productare examined for the expression of essential genes that stimulatefunctionally active chondrocytes. Substantial expression levels of thefollowing are detected: collagen type II, aggrecan, SOX5, SOX6, andSOX9.

Example 25 The Stimulatory Effect of Chondrocytes in Cartilage Productson Exogenous MSCs

Isolated mesenchymal stem cells (e.g. from a different donor) areco-cultured with chondrocytes isolated from cartilage products of thepresent invention.

The chondrocytes stimulate MSCs to differentiate to chondrocytes andstimulate the resultant chondrogenesis in this model. These resultsdemonstrate that therapeutic efficacy of cartilage products of thepresent invention is due, in part, to the stimulatory effect ofchondrocytes of the cartilage product on recipient MSC cells.

Example 26 Spiral Cut Cartilage Products

Cartilage products are produced according to the method detailed inExample 9 except that, rather than porated, the cartilage samples arespiral cut with either removal of tissue (e.g. as depicted in FIG. 20 orFIG. 21) or without removal of tissue (e.g. as detailed in FIG. 17C).Additional cartilage products are produced using the same method exceptthat the step of partial digestion is eliminated.

The spiral cut cartilage products are flexible and can conform to atarget site in a patient and can easily be administered via arthroscope.

The spiral cut cartilage products show efficacy in a model of chondraldefects, as detailed in Example 22.

After a period of time, the implanted spiral cut cartilage products andsurrounding host tissue are removed and analyzed, and using appropriatemarkers, one or more of the following is observed:

-   -   a. integration into host cartilage    -   b. infiltration of host cells and ECM rich in type II collagen;    -   c. substantial expression of bioactive factors such as        chondrogenic factors    -   d. substantial levels chondrocytes    -   e. substantial expression levels of collagen type II, aggrecan,        SOX5, SOX6, and SOX9.

The spiral cut cartilage products show superior efficacy when treatingsubjects (e.g. cartilage defects). Due, in part, to the enhancedflexibility provided by the spiral cut, larger (e.g. greater thickness,greater area, or greater volume) cartilage products can be produced thatcan be easily manipulated and conform to a target site (e.g. voidremaining after removal of a cartilage defect). Due, in part, to theconfiguration discontinuities (spiral cut), the cartilage product canexpand or contract (e.g. laterally) in vivo to provide enhancedintegration into host cartilage.

Example 27 Mechanically Disrupted Cartilage Products

Cartilage products are produced according to the method detailed inExample 9 except that, rather than porated, the cartilage samples aremechanically disrupted using aligned cuts, radial cuts, intersectingcuts, hoop-forming cuts, or multiple hoop-forming cuts. Each type ofmechanical disruption was performed at least twice on differentcartilage samples, once with removal of tissue (e.g. as depicted in FIG.18 and FIG. 19) and once without removal of tissue (e.g. as detailed inFIG. 17). Additional cartilage products are produced using the samemethod except that the step of partial digestion is eliminated.

The mechanically disrupted cartilage products have the followingsuperior properties, which are present to even a greater extent in thepartially-digested products.

The mechanically disrupted cartilage products are flexible and canconform to a target site in a patient and can easily be administered viaarthroscope.

The mechanically disrupted cartilage products show efficacy in a modelof chondral defects, as detailed in Example 22.

After a period of time, the implanted mechanically disrupted cartilageproducts and surrounding host tissue are removed and analyzed, and usingappropriate markers, one or more of the following is observed:

-   -   a. integration into host cartilage    -   b. infiltration of host cells and ECM rich in type II collagen;    -   c. substantial expression of bioactive factors such as        chondrogenic factors    -   d. substantial levels chondrocytes    -   e. substantial expression levels of collagen type II, aggrecan,        SOX5, SOX6, and SOX9.

The citations provided herein are hereby incorporated by reference forthe cited subject matter.

Further embodiments of the present invention can be found in thefollowing paragraphs.

In some embodiments, the present technology provides a cartilage productcomprising a disrupted collagen matrix.

In some embodiments, the cartilage product contains collagen matrixcomprises enzymatic disruptions.

In some embodiments, the collagen matrix is collagenase-digested,optionally wherein the collagen matrix is collagenase II-digested.

In some embodiments, the present technology provides a cartilage productwherein the collagen matrix comprises mechanical disruptions. In someembodiments, the mechanical disruptions are partial mechanicaldisruptions, wherein the collagen matrix is substantially intact. In yetother embodiments, the mechanical disruptions are selected from: spiralcut, grooves, scores, fenestrations, and pores. In further embodiments,the mechanical disruptions may be full mechanical disruptions,optionally wherein the collagen matrix minced. In some embodiments, themechanical disruptions are of the tissue-removal type. In anotherembodiment, the mechanical disruptions are of the tissue-non-removaltype.

In yet another embodiment, the cartilage product includes a collagenmatrix comprising mechanical disruptions, wherein the mechanicaldisruptions extend through the entire thickness of the collagen matrix.In yet another embodiment, the mechanical disruptions are selected from:spiral cut, scores, intersecting scores, radial scores, hoop-cut, andpores.

In other embodiments, the cartilage product contain collagen matrixcomprising mechanical disruptions, wherein the mechanical disruptionsextend through less than the entire thickness of the collagen matrix. Insome embodiments, the mechanical disruptions are selected from: spiralcut, scores, intersecting-scores, and radial scores.

In some embodiments, the cartilage product of the present technologyincludes a collagen matrix has a thickness of about 0.2 mm to about 2.0mm, alternatively a thickness of about 1 mm to about 1.5 mm.

In yet another embodiment, the present technology provides a cartilageproduct wherein the collagen matrix contains viable cells, optionallywherein the viable cells are native to the collagen matrix. In yetanother embodiments, the cartilage product of the present technologyincludes a collagen matrix is decellularized.

In some embodiments, the cartilage product of the present technologyfurther comprising a layer of bone, optionally wherein the layer of boneis native to the collagen matrix. In some embodiments, the layer of bonehas a surface area substantially less than that of the collagen matrix.In yet another embodiment, the layer of bone is a plug, a shelled plug,or a shelled layer or bone.

In some embodiments, the cartilage product of the present technologycontains collagen matrix that is a natural human collagen matrix. In yetanother embodiment, the collagen matrix comprises hyaline cartilage.

In yet another embodiment, the cartilage product of the presenttechnology comprises hyaline cartilage. In some embodiments, the hyalinecartilage comprises articular cartilage.

In yet another embodiment, the cartilage product of the presenttechnology contains a collagen matrix, wherein the collagen matrixcomprises viable chondrocytes in an amount of at least any of: about 500cells/mm², about 600 cells/mm², about 700 cells/mm², about 800cells/mm², about 1200 cells/mm², about 1500 cells/mm², and about 5000cells/mm², optionally wherein the viable chondrocytes are native to thecollagen matrix.

In yet another embodiment, the cartilage product of the presenttechnology contains a collagen matrix, wherein the collagen matrixcomprises one or more layers selected from: a tangential layer, atransitional layer, and a radial layer. In some embodiments, thecollagen matrix comprises a tangential layer comprising at least any of:about 100 cells/mm², about 200 cells/mm², and about 1000 cells/mm²,optionally wherein the viable chondrocytes are native to the collagenmatrix.

In some embodiments, the cartilage product contains a collagen matrixcomprising a transitional layer comprising at least any of: about 100cells/mm², about 200 cells/mm², and about 400 cells/mm², optionallywherein the viable chondrocytes are native to the collagen matrix.

In other embodiments, the cartilage product contains a collagen matrixcomprising a radial layer comprising at least any of: about 100cells/mm² and about 200 cells/mm², optionally wherein the viablechondrocytes are native to the collagen matrix.

In another embodiment, the cartilage product of the present technologycontains a collagen matrix, wherein the collagen matrix is flexible,optionally wherein the collagen matrix has a flexibility such that itcan be bend or folded without breaking. In some embodiments, thecartilage product contains collagen matrix that has a Young's modulus ofat least about any of: 0.1 MPa, 0.2 MPa, 0.3 MPa, and 0.4 MPa.

Another embodiment provides method of treatment comprising administeringto a subject in need thereof, a cartilage product of the presenttechnology. In some embodiments, the cartilage product is administeredarthroscopically. In another embodiment, administering comprises foldingor rolling the cartilage product. In yet another embodiment, the step ofadministering is performed in conjunction with a microfractureprocedure.

In another embodiment, the cartilage product of the present technologyincludes collagen matrix comprising type II collagen. In someembodiments, the cartilage product includes collagen matrix comprisinghyaline cartilage. In some embodiments, the cartilage product includescollagen matrix that is a natural collagen matrix, optionally, whereinthe collagen matrix is a human collagen matrix.

In yet another embodiment, the cartilage product of the presenttechnology includes collagen matrix that is a natural collagen matrix,optionally, wherein the collagen matrix is a human collagen matrix.

In some embodiments, the cartilage product further comprising anadditive.

What is claimed is:
 1. A cartilage product comprising a disruptedcollagen matrix.
 2. The cartilage product of claim 1, wherein thecollagen matrix comprises enzymatic disruptions.
 3. The cartilageproduct of claim 2, wherein the collagen matrix is collagenase-digested,optionally wherein the collagen matrix is collagenase II-digested. 4.The cartilage product of claim 1, wherein the collagen matrix comprisesmechanical disruptions.
 5. The cartilage product of claim 4, wherein themechanical disruptions are partial mechanical disruptions, wherein thecollagen matrix is substantially intact.
 6. The cartilage product ofclaim 5, wherein the mechanical disruptions are selected from: spiralcut, grooves, scores, fenestrations, and pores.
 7. The cartilage productof claim 4, wherein the mechanical disruptions are full mechanicaldisruptions, optionally wherein the collagen matrix minced.
 8. Thecartilage product of claim 4, wherein the mechanical disruptions are ofthe tissue-removal type.
 9. The cartilage product of claim 4, whereinthe mechanical disruptions are of the tissue-non-removal type.
 10. Thecartilage product of claim 8, wherein the mechanical disruptions extendthrough the entire thickness of the collagen matrix.
 11. The cartilageproduct of claim 10, wherein the mechanical disruptions are selectedfrom: spiral cut, scores, intersecting scores, radial scores, hoop-cut,and pores.
 12. The cartilage product of claim 8, wherein the mechanicaldisruptions extend through less than the entire thickness of thecollagen matrix.
 13. The cartilage product of claim 12, wherein themechanical disruptions are selected from: spiral cut, scores,intersecting-scores, and radial scores.
 14. The cartilage product ofclaim 1, wherein the collagen matrix has a thickness of about 0.2 mm toabout 2.0 mm.
 15. The cartilage product of claim 14, wherein thecollagen matrix has a thickness of about 1 mm to about 1.5 mm.
 16. Thecartilage product of claim 1, wherein the collagen matrix containsviable cells, optionally wherein the viable cells are native to thecollagen matrix.
 17. The cartilage product of claim 1, wherein thecollagen matrix is decellularized.
 18. The cartilage product of claim15, further comprising a layer of bone, optionally wherein the layer ofbone is native to the collagen matrix.
 19. The cartilage product ofclaim 18, wherein the layer of bone has a surface area substantiallyless than that of the collagen matrix.
 20. The cartilage product ofclaim 19, wherein the layer of bone is a plug, a shelled plug, or ashelled layer or bone.